1. Field of the Invention
Microbial enzymes are becoming increasingly important in such diverse fields as medicine, agriculture and organic chemicals production. The genus Bacillus comprises a group of bacteria that can be easily maintained and cultivated, yet are markedly heterogeneous in character. All of the 48 species of the Bacillus genus listed in Bergey's Manual of Determinative Bacteriology secrete soluble enzymes.
The microorganism B. subtilis offers numerous advantages for industrial processes and has been used for decades in industrial fermentations that yield amylase, proteases and other products.
Some of the advantages of B. subtilis include the production of various proteins (extracellular) which are completely dissociated from the cell and found free in the surrounding medium, raising the possibility of altering such microorganism to produce commercial fermentation products. In addition, B. subtilis does not survive in or on vertebrates and has not been shown to produce any serious human illness.
It is well known that the genetic information of all cells is stored in deoxyribonucleic acid (DNA) in the chromosomal material of microorganisms. The unit of genetic function, i.e., the locus on the chromosome related to a specific hereditary trait, is called a gene.
Prior to the advent of recombinant DNA technology, gene studies and genetic manipulations have been carried out by the classical genetic techniques of transformation and transduction. Recombinant DNA technology involves the transfer of genetic material (genes, or DNA fragments) from one organism into a second organism, by means of a transfer component designated a "vector", producing a combination of genetic material. The second organism (which contains the transferred genetic material) is designated a recombinant component. The recombinant component can then be used as a source of DNA to insert genetic material into bacterial and animal cells for propagation of the combined genes contained in the recombinant component. The cell into which the DNA of the recombinant component is inserted is designated a host cell.
Using recombinant DNA technology, genetic modification can be accomplished as follows. Specific DNA fragments from a vector, e.g., a lysogenic bacteriophage are "isolated", e.g., by treatment with appropriate restriction enzymes which act as "chemical scalpels" to split DNA molecules into specific fragments which usually contain from less than 1 to 10 genes each, or by other well known techniques. A DNA fragment for the desired genetic characteristic, i.e., "foreign DNA" from the bacterial source is then inserted into the DNA bacteriophage vector. By treatment with DNA ligase the DNA fragment is inserted into the bacteriophage DNA vector and a recombinant bacteriophage DNA molecule is formed. The recombinant bacteriophage contains all or most of the genes of the bacteriophage plus the new genes (foreign DNA) from the inserted fragment. This recombinant bacteriophage can be introduced into a host bacterium thereby "cloning" the foreign DNA into the host. The new genes are propagated and become a part of the genetic machinery of the bacterium host. If successful, the bacterium host thus acquires the gentic traits contributed by the new genes and is capable of "expressing" these traits.
2. Prior Art
Previous studies have established that the level of .alpha.-amylase synthesis in B. subtilis is regulated by a number of genes. Studies have indicated that .alpha.-amylase is regulated by a specific regulatory gene (amyR) that can be linked to its structural gene (amyE) by transformation. [See J. Bacteriol. 119: 410-415 (1974); J. Gen. Appl. Microbiol., 15: 97-107 (1969) and Biochem. Biophys. Res. Commun., 31: 182-187 (1968)]. In addition to production of .alpha.-amylase attributed to the amyE and amyR genes, .alpha.-amylase production can be achieved by a "pleiotrophic" mutation, i.e., by a gene that regulates more than one function. Known pleiotrophic mutations include the papM gene ("production of .alpha.-amylase and protease") and a gene designated tmr encoding resistance to the antiviral antibiotic tunicamycin; both these genes are regulatory genes.
It is known that genes according or regulating .alpha.-amylase in a Bacillus strain can be introduced into B. subtilis if the two strains are sufficiently closely related, i.e., if there is extensive genetic homology between the two strains. This is referred to as homologous transformation. For example, J. Bacteriol., 120: 1144-1150 (1974) describes the introduction of DNA from B. subtilis var amylosacchariticus having exceptionally high .alpha.-amylase activity, into a genetically similar (homologous) microorganism having relatively low .alpha.-amylase activity (B. subtilis Marburg). The transformed microorganisms which were produced acquired high .alpha.-amylase activity.
However, most Bacillus are not sufficiently related to B. subtilis, i.e., are not sufficiently homologous, to permit the DNA obtained from one Bacillus subtilis strain to be efficiently introduced into a different Bacillus strain. J. Bacteriol., 111: 705-716 (1972).
Appl. Environ. Microbiol. 39: 274-276 (1980) established that the effect of incorporating the related genes (amyR3, amyS, papS1, tmr and papM118) into a strain produced an increase in .alpha.-amylase production of a synergistic nature. The overall approach involved the stepwise introduction of the amy, pap and tmr genes into a recipient B. subtilis Marburg 6160 (a B. subtilis 168) microorganism by a stepwise transformation procedure. The authors indicate that because the transformation procedure requires chromosomal homology, a suggested alternative approach which can utilize chromosomal heterology would involve the development of vectors for cloning the genes and introducing them into a modified recipient, i.e., a "mother cell" in a more purified form.
Although the regulatory genes from Bacillus natto and Bacillus subtilis var. amylosacchariticus can be introduced readily into homologous B. subtilis 168 by DNA-mediated transformation, it is extraordinarily difficult to use such conventional transformation techniques in a heterologous transformation, e.g., to introduce genes from B. amyloliquefaciens into B. subtilis 168. As indicated in J. Bacteriol., 111: 705-716 (1972), particularly Table 2, transformation of a B. subtilis 168 strain (BR151) with DNA from a homologous B. subtilis 168 strain produced one thousand-fold more transformants for three tested loci than DNA from a heterologous source, B. amyloliquefaciens H.
Biochem. Biophys. Res. Comm., 91: 1556-1564 (1979) describes a method of cloning heterologous genes in bacteriophage .phi.3T, producing a specialized transducing bacteriophage containing the genetic information encoding .alpha.-amylase from B. amyloliquefaciens H.
However, while the above paper predicts that the use of such a technique will allow the insertion of a variety of Bacillus genes encoding extracellular enzymes into B. subtilis it is not predictable whether regulatory genes inserted into the B. subtilis will also function to regulate or enhance the production of .alpha.-amylase encoded by a foreign (i.e., heterologous) cloned gene.